Cellulose is the main component of plant cell wall, and is a renewable carbon source having the largest amount on the earth. The use of cellulose in the production of fuel ethanol and other chemicals via bioconversion is of extremely important practical significance in the resolution of the energy crisis, food shortages, environmental pollution and other challenges to human beings.
Cellulase, which degrades cellulose, is a multi-enzyme composite system, comprising endoglucanase, exo-cellobiohydrolase and β-glucosidase. These three enzymes act synergistically to degrade cellulose into glucose. Among others, β-glucosidase (EC 3.2.1.21, abbreviated as BGL), could hydrolyze cellobiose to release glucose during the hydrolysis process of cellulose by cellulase, which constitutes the key step of the complete hydrolysis of cellulose.
Generally, there are several issues in the cellulose hydrolysis process as follows. Firstly, the activity of β-glucosidase is inhibited by the hydrolysis product, i.e., glucose, while cellobiose is an inhibitor of the other enzymes in the cellulase system, leading to the inhibition of the whole hydrolysis process of cellulose. Secondly, in cellulase-producing strains, β-glucosidase content is very low (except Aspergillus niger). For example, among the cellulases secreted by T. reesei, the β-glucosidase content is less than 1%, far less than the level for practical application. Thirdly, the thermal inhibition renders significant loss of β-glucosidase activity during the reaction process. As a result, the activity of β-glucosidase is generally lower than that of endoglucanase and exoglucanase by an order of magnitude or more, becoming the rate-limiting enzyme of cellulose degradation (Wang Zhenyu, et. al., “The activity of the endocellulase and β-glucosidase produced by high temperature anaerobic organism”, Journal of Dalian Institutes of Light Industry, 2005, 24 (2), 110-114).
Currently, the most widely used cellulase-producing strains are mostly superior mutant strains of Trichoderma reesei, which can secret and produce endoglucanase, exoglucanase and β-glucosidase, constituting the whole enzyme system for cellulose hydrolysis (Penttila M. E. et. al., “Expression of two Trichoderma reesei Endoglucanase in the Yeast Saccharomyces cereviae”, Yeast. Vol. 3, 1987(3): 175-185).
Due to low β-glucosidase production in natural T. reesei strains, the hydrolysis process of cellulose is limited. Therefore, the improvement of β-glucosidase activity in the cellulase system is one of the key measures to improve the yield of cellulase hydrolysis and glucose production.
Some researchers subjected T. reesei β-glucosidase gene to recombinant expression in Pichia yeasts, and found that the enzyme activity increases along with the duration of induction time, but the fermentation period thereof is up to more than 8 days (Chinese Patent CN102220369B; “Recombinant vector of Trichoderma reesei beta-glucosidase gene BGL1, recombinant microorganism, and expression of BGL1 in the recombinant microorganism”). Some researchers over-expressed β-glucosidase in T. reesei by fusion expression, and achieved enzyme activity several times higher than that of the original strain, but the fermentation period was up to more than 6 days (Chinese Patent CN 101516906B; “Methods for increasing secretion of polypeptides having biological activity”). Generally, the existing methods are restricted by the disadvantage of overlong fermentation periods.
Therefore, there is an urgent need in the art to develop a cellulose degrading microorganism (for example, genetically engineered T. reesei) capable of improving the enzyme activity while shortening the fermentation period, thereby greatly reducing production costs and improving the competitiveness of the cellulase.